Life 2014, 4, 77-104; doi:10.3390/life4010077 OPEN ACCESS
life ISSN 2075-1729 www.mdpi.com/journal/life Review
Anaerobic Thermophiles Francesco Canganella 1,* and Juergen Wiegel 2 1
2
Department for Innovation in Biological, Agrofood, and Forest Systems, University of Tuscia, via C. de Lellis, Viterbo 01100, Italy Department of Microbiology, University of Athens (GA), Athens 10679, USA; E-Mail:
[email protected]
* Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +39-0761-357282. Received: 3 June 2013; in revised form: 10 January 2014 / Accepted: 26 January 2014 / Published: 26 February 2014
Abstract: The term ―extremophile‖ was introduced to describe any organism capable of living and growing under extreme conditions. With the further development of studies on microbial ecology and taxonomy, a variety of ―extreme‖ environments have been found and an increasing number of extremophiles are being described. Extremophiles have also been investigated as far as regarding the search for life on other planets and even evaluating the hypothesis that life on Earth originally came from space. The first extreme environments to be largely investigated were those characterized by elevated temperatures. The naturally ―hot environments‖ on Earth range from solar heated surface soils and water with temperatures up to 65 °C, subterranean sites such as oil reserves and terrestrial geothermal with temperatures ranging from slightly above ambient to above 100 °C, to submarine hydrothermal systems with temperatures exceeding 300 °C. There are also human-made environments with elevated temperatures such as compost piles, slag heaps, industrial processes and water heaters. Thermophilic anaerobic microorganisms have been known for a long time, but scientists have often resisted the belief that some organisms do not only survive at high temperatures, but actually thrive under those hot conditions. They are perhaps one of the most interesting varieties of extremophilic organisms. These microorganisms can thrive at temperatures over 50 °C and, based on their optimal temperature, anaerobic thermophiles can be subdivided into three main groups: thermophiles with an optimal temperature between 50 °C and 64 °C and a maximum at 70 °C, extreme thermophiles with an optimal temperature between 65 °C and 80 °C, and finally hyperthermophiles with an optimal temperature above 80 °C and a maximum
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above 90 °C. The finding of novel extremely thermophilic and hyperthermophilic anaerobic bacteria in recent years, and the fact that a large fraction of them belong to the Archaea has definitely made this area of investigation more exciting. Particularly fascinating are their structural and physiological features allowing them to withstand extremely selective environmental conditions. These properties are often due to specific biomolecules (DNA, lipids, enzymes, osmolites, etc.) that have been studied for years as novel sources for biotechnological applications. In some cases (DNA-polymerase, thermostable enzymes), the search and applications successful exceeded preliminary expectations, but certainly further exploitations are still needed. Keywords: anaerobic thermophiles; thermal ecosystems; extremophiles; deep-sea; taxonomy; biotechnology
1. Introduction Among anaerobic and thermophilic microorganisms, anaerobic thermophilic Archaea are certainly the most ―extreme‖ in terms of inhabited ecosystems. They represent the deepest, least evolved branches of the universal phylogenetic tree (Figure 1). They often use substrates, which are thought to have been dominant in the primordial terrestrial makeup, indicating that they could have been the first living forms on this planet [1–6]. Studies into how they manage thermostability at the protein and membrane structural level have elucidated many traits of protein, membrane and nucleic acid structure; however, there is not yet a full understanding of the principles of thermostability [7–11]. The development of better genetic tools for the use of these organisms is the key for more practical applications in the future [12–14]. Figure 1. Phylogenetic tree highlighting possible evolutionary relatedness of anaerobic thermophilic Archaea (modified from Eric Gaba, NASA Astrobiology Institute 2006).
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Although the first forms of life no longer exist, natural thermal environments are still abundant on Earth and some have properties similar to those environments in which life possibly first began. Many of these environments are characteristically anaerobic or have low levels of oxygen. The anaerobic feature can stem from a number of factors: remoteness of the environment from the atmosphere, low solubility of oxygen in water at elevated temperatures, hypersalinity, inputs of reducing gasses such as H2S, or the consumption of oxygen by aerobic microorganisms on or near the water surface. Natural environments for anaerobic thermophiles range from terrestrial volcanic sites (including solfatara fields) with temperatures slightly above ambient temperature, to submarine hydrothermal systems (sediments, submarine volcanoes, fumaroles and vents) with temperatures exceeding 300 °C, subterranean sites such as oil reservoirs, and solar heated surface soils with temperatures up to 65 °C (Figures 2 and 3). There are also human-made hot environments such as compost piles (usually around 60–70 °C but as high as 100 °C) slag heaps, industrial processes and water heaters [15]. Figure 2. Some environments where anaerobic thermophiles can be isolated: (a) A power plant in Iceland; (b) Terrestrial hot springs at Viterbo (Italy); (c) The hot pool of Bagno Vignoni (Italy).
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Figure 3. Deep-sea hot ecosystems: (a) Hot sediment at the Guaymas Basin; (b,c) Drawings of black smokers located at a deep-sea hydrothermal vent area (courtesy of Focus Magazine and Jack Jones, respectively).
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Oil reservoirs, mines, and geothermal aquifers are examples of subsurface environments that thermophiles populate. Extreme thermophilic bacterial species of the genera Geotoga and Petrotoga (family Thermotogaceae) have so far only been found in deep subsurface oil reservoirs; on this basis, it has been proposed that these taxa represent typical indigenous Bacteria of this particular ecosystem. However, lately Thermotogales sequences have been found in mesobiotic environments [16] and novel species have been described [17]. Geothermal aquifers, such as the Great Artesian Basin of Australia, are considered to be markedly different from volcanically related hot springs in that they have low flow rates and long recharge times (around 1000 years) that affect the microbial populations therein. Besides natural thermal environments, thermophilic anaerobes are also found within anthropogenically
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heated environments, including coal refuse piles and compost heaps, and nuclear power plant effluent channels which contain not only spore-forming species, but also vegetative and active cells including Bacteria and Archaea. Many environments are also temporarily hot, adaptation to which may be the reason some thermophiles are very fast-growing. Among the geothermally heated habitats are the alkaline, mainly carbonate-containing hot springs around a neutral pH, and acidic areas including some mud-holes. Most of the acidic high-temperature habitats contain elemental sulfur and metal sulfides and most isolates from these areas metabolize sulfur by either anaerobic respiration or fermentation. Ocean depths are under extreme pressures from the weight of the water column, and thus most anaerobic and thermophilic bacteria from these areas are piezotolerant, some are truly piezophilic, others such as Pyrococcus spp., Thermococcus spp., and M. kandleri show extensions of Tmax under increased pressure [18–23] and all are at least halotolerant [24], and those isolated from solfataras generally acidophilic. Alike most described species of obligately aerobic thermophilic Archaea that are acidophilic, anaerobic thermophilic bacteria are generally unable to grow at acidic pH with some exemptions such as representatives of genera Stygiolobus, Acidilobus, and Caldisphaera [25–27]. On the other hand many anaerobic bacteria and some Archaea are capable of growing at an alkaline pH [28]. The anaerobic alkalithermophilic bacteria thus form an interesting group to study, and their relationships between temperature optimum and pH optimum for growth have been extensively investigated. This adaptability to high pH environments involves both cellular and biomolecular peculiar traits that are currently under investigation, particularly to exploit their potential biotechnological applications. Among extreme environments, the deep sea is in general cold, but it is known to show areas of superheated water and widespread still-hot volcanic ocean crust beneath the flanks of the mid-ocean ridge and other rock structures, as well as geothermally heated shallower ocean waters. A large group of anaerobic and thermophilic microorganisms have been isolated and studied from the deep-sea, particularly at both hydrothermal vents and sub-seafloor sites, either for their physiological properties or for their potential applications [29–34]. Representative deep-sea environments, if not in terms of geographical extension but certainly as the most spectacular, are the deep-sea hydrothermal vents. The highly dense and biologically diverse communities in the immediate vicinity of hydrothermal vent flows are in stark contrast to the surrounding bare seafloor. They comprise organisms with distinct metabolisms based on chemosynthesis and growth rates comparable to those from shallow water tropical environments, which have been rich sources of biologically active natural products. Fundamental discoveries in this regard will be accelerated by new cost-effective technologies in deep-sea research and more advanced molecular techniques. Taxonomical and phylogenetic investigations have always been the main focus concerning research on deep-sea anaerobic thermophiles. Diversity and richness of deep-sea hydrothermal environments were particularly examined and shown to be as high as those in soil. As a matter of fact, sediments from deep-sea floors have always been great sources of novel bacterial isolates and recently new genera as well as species are being described from different sites in the ocean depths [35–37]. As far as concerns the diversity of sub-seafloor microorganisms, a ―meta-enzyme approach‖ has been proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine habitats [38]. Detectable enzyme
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activities were used to predict the existence of a sizable population of viable anaerobic microorganisms even in deep sub-seafloor habitats. Moreover many microbial isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases, giving them a great potential in terms of biotechnological applications. A main topic in ecology and population dynamics of deep-sea anaerobic thermophiles is their colonization and distribution patterns along and around hydrothermal vent deposits. An approach based on the deployment of thermocouple arrays on two deep-sea hydrothermal vents at Guaymas Basin was adopted by Pagé et al. [39]. This aimed to measure in situ temperatures at which microorganisms colonize the associated mineral deposits. Spatial differences in archaeal diversity were observed in all deposits in relationship to in situ temperature. This study was the first direct assessment of in situ conditions experienced by microorganisms inhabiting actively forming hydrothermal deposits at different stages of structure development. 2. Growth Conditions Microorganisms that grow optimally at elevated temperatures above 50 °C and can not use oxygen as terminal electron acceptor during electron transport phosphorylation are described as thermophilic anaerobes. They are of interest from basic and applied scientific perspectives and are studied to understand how life can thrive in environments previously considered inhospitable to life. Such environments include volcanic solfataras and hot springs high in sulfur and toxic metals, as well as abyssal hydrothermal vents with extremely high pressure and temperatures far above 100 °C [40]. Isolated species of thermophilic anaerobes include peculiar forms: for example, cells of the alkalithermophile Clostridium paradoxum become highly motile when sporulating, and Moorella thermacetica-like strains have exceptionally heat-resistant spores with D10 times of nearly 2 h at 121 °C. Also, Pyrolobus fumarii grows optimally at 106 °C, and the record-holder Methanopyrus kandleri-like strain grows at 122 °C under increased pressure [18]. Thermobrachium celere strains have doubling times of about 10 min while growing above pH 9.0 and above 55 °C [41] and the polyextremophilic Natranaerobius isolates simultaneously grow optimally up to 69 °C and above pH 9.5, and at a salt concentration above 4 M Na+. They may be considered the most extremophile as they withstand the combination of multiple stressors. It will be of interest to evaluate whether those boundaries can be further extended by isolating other polyextremophiles [42–44]. The analyses of the biodiversity and patterns of biodiversity within thermal environments is an area of active research that continually expands as technology allows for novel approaches and more detailed analyses. Additionally, their thermostable enzymes, among other characteristics, make thermophilic anaerobes of significant interest for their biotechnological potential. Contrary to any expectation, thermophilic anaerobes have also been isolated from mesobiotic and even psychrobiotic environments: two Thermosediminibacter species were isolated from ocean sediments of the Peru Margin at temperatures at or below 12 °C [45], uncharacterized Thermoanaerobacter species have been isolated from melted snow from Antarctica (unpublished results), alkalithermophiles have been isolated from many river sediments and wet meadows, and Methanothermobacter thermoautotrophicus and other thermophilic methanogens and chemolithoautotrophic acetogens can readily be found in lake sediments and rivers, streams, and
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ponds. Possible reasons for the presence of thermophilic anaerobes in environments where they were thought not to grow, considering their physiological properties, include (a) that the microorganisms are present but do not grow in these environments although they are able to carry out maintenance metabolism (e.g., as shown for M. thermoautotrophicum that is able to form methane at temperatures as low as 16 °C, although it is not able to multiply at temperatures below 22 °C (Wiegel unpl. results); (b) that they disperse only transiently from other thermal environments; (c) that they survive and multiply by taking advantage of temporary thermal piconiches that become available when proteinaceous biomass is degraded. The latter notion is further substantiated by observations that strains of Calaromator (Bas. Thermobrachium) celer isolated from mesobiotic environments show very short doubling times (between 10 min and 20 min), whereas the strains of the same species isolated from hot springs—which resemble a more constant thermobiotic environment—have doubling times of above 30 min (Wiegel, unpublished results) and also that the moderate thermophiles C. paradoxum- and the nonsporulating C. thermophilus-like cells are present in mesobiotic sewage sludge (
